Simvastatin Spleen Atropi

NIH Public Access
Author Manuscript
Stroke. Author manuscript; available in PMC 2014 April 01.
NIH-PA Author Manuscript
Published in final edited form as:

Stroke. 2013 April ; 44(4): 11351143. doi:10.1161/STROKEAHA.111.000633.
Simvastatin attenuates stroke-induced splenic atrophy and lung

susceptibility to spontaneous bacterial infection in mice
Rong Jin, MD, PhD, Xiaolei Zhu, MD, PhD, Lin Liu, MD, PhD, Anil Nanda, MD, D Neil
Granger, PhD, and Guohong Li, MD, PhD
Vascular Biology and Stroke Research Laboratory of Department of Neurosurgery (J.R., Z.X.,
L.L., N.A., L.G.) and Department of Physiology (D.N.G., L.G.), LSU Health Science Center in
Shreveport, Shreveport, Louisiana, United States of America
Abstract
Background and PurposeStatins are widely used in the primary and secondary prevention
of ischemic stroke, but their effects on stroke-induced immunodeppression and post-stroke
infections are elusive. We investigated effects of simvastatin treatment on stroke-induced splenic
atrophy and lung susceptibility to bacterial infection in acute experimental stroke in mice.
MethodsIschemic stroke was induced by transient occlusion of middle cerebral artery
(MCAO) followed by reperfusion. In some experiments, splenectomies were performed 2 weeks
prior to MCAO. Animals were randomly assigned to sham and MCAO groups treated
subcutaneously with vehicle or simvastatin (20 mg/kg/day). Brain infarction, neurological
function, brain interferon- expression, splenic atrophy and apoptosis, and lung infection were
examined.
ResultsSimvastatin reduced stroke-induced spleen atrophy and splenic apoptosis via increased
mitochrondrial anti-apoptotic Bcl-2 expression and decreased pro-apoptotic Bax translocation
from cytosol into mitochondria. Splenectomy reduced brain interferon- (3d) and infarct size (5d)
after stroke and these effects were reversed by adoptive transfer of splenocytes. Simvastatin
inhibited brain interferon- (3d) and reduced infarct volume and neurological deficits (5d) after
stroke, and these protective effects were observed not only in nave stroke mice but also in
splenectomied stroke mice adoptively transferred with splenocytes. Simvastatin also decreased the
stroke-associated lung susceptibility to spontaneous bacterial infection.
ConclusionsResults provide the first direct experimental evidence that simvastatin

ameliorates stroke-induced peripheral immunodepression by attenuating spleen atrophy and lung
bacterial infection. These findings contribute to a better understanding of beneficial effects of
statins in the treatment of stroke.
Keywords
Brain ischemia; bacterial infection; immune response; statin; spleen
Correspondence to Dr. Guohong Li, MD., PhD, the Vascular Biology and Stroke Research Laboratory, Department of Neurosurgery,
Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130. gli@lsuhsc.edu.
First two authors contributed equally to this work.
Disclosures
None.
Jin et al.
Page 2
Introduction
Stroke is the third leading cause of death in the world, but treatment options for acute stroke
remain limited. Cerebral ischemia not only produces local brain damage, but also triggers a
profound systemic immunodepression or stroke-induced immunodeficiency syndrome,
which predisposes patients after stroke to infections [1-3]. Infectious complications,
predominantly chest and urinary tract infections, occur in many stroke patients within the
first few days after stroke, and are a leading cause of death in patients with acute stroke
[4,5]. The prognosis of stroke depends strongly on the incidence of infectious complications,
in particular pneumonia. Pneumonia is the most common post-stroke infection, about half of
pneumonia cases occur within the first 2 days after stroke onset, and almost all cases occur
within the first week [4, 5]. Thus, therapeutic modification of post-stroke immunodepression
and avoiding poststroke infections may represent a valuable approach to improving outcome
in stroke patients [6].
Statins are inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)

and they are the most effective agents for lowering cholesterol in clinical practice for the
treatment of cardiovascular diseases. Recently, it has become clear that statins also have
pleiotropic effects on inflammation and immunity in addition to their lipid-lowering
properties [7]. Previous studies have reported that statins have beneficial effects on various
forms of experimental brain injury such as ischemic stroke [8,9], subarachnoid hemorrhage
[10], and intracerebral hemorrhage [11,12]. Statins are widely used in the primary and
secondary prevention of ischemic stroke in patients [13]. Pretreatment of experimental
stroke with simvastatin significantly reduces brain infarct volume and improves neurological
outcome [14]. Delayed treatment of experimental stroke with a statin starting 24 hours after
stroke does not decrease lesion volume, but significantly improves functional outcome
compared to animals with nontreated stroke [15, 16].
Post-stroke peripheral immunodepression is characterized by profound atrophy of secondary

lymphatic organs (spleen, thymus and lymph node) that occur within the first few days after
stroke [1, 2]. Several recent studies have reported that spleen contributes to
neuroinflammation and the development of ischemic stroke in acute experimental stroke
[17-21]. Splenectomy two weeks prior to middle cerebral artery occlusion (MCAO) in the
rat significantly reduces neuroinflammation and brain damage [22]. To our knowledge, no
experimental studies have addressed if and how statins affect stroke-induced
immunodeppression and post-stroke infections. Thus, the present study was designed to
investigate the effects of simvastatin on stroke-induced peripheral immunodeppression and
post-stroke infections. We focused on investigation on the effects of simvastatin on strokeinduced splenic atrophy and lung susceptibility to bacterial infection.
Methods
Ethics Statement
All experimental procedures were carried out in accordance with the NIH Guide for the Care
and Use of Laboratory Animals and approved by the Animal Care and Use Committee of the
Louisiana State University Health Science Center-Shreveport (IACUC approval number:
11010). Male C57BL/6J mice (8 to10 weeks old, 22 to 25g), obtained from Jackson
Laboratories (Bar Harbor, Me), were used in the study.
Experimental Model of Stroke
Focal cerebral ischemia was induced by transient occlusion of the left middle cerebral artery
(MCAO) with a 6-0 silicone-coated nylon monofilament (Doccol Corp.), as previously
described [23]. Subgroups of mice were randomly selected to receive MCAO or sham
Jin et al.
Page 3
procedures. In ischemic groups, animals were subjected to 60 minutes MCAO followed by

reperfusion for indicated times. In the sham controls, the arteries were visualized but not
disturbed. Rectal temperature was maintained at 370.5C throughout the procedure from
the start of the surgery until the animals recovered from anesthesia with a feed-back
regulated heating pad. Regional cerebral blood flow (rCBF) was monitored by laser Doppler
flowmetry (MSP300XP; ADInstruments Inc) as previously described [23]. Only animals
that exhibited a reduction in CBF>85% during MCAO and a CBF recovery by >80% after
15 min of reperfusion were included in the study.
Splenectomy and Adoptive Transfer of Splenocytes
In some experiments, splenectomy was performed two weeks prior to MCAO using the
published procedure [24]. Briefly, under anesthesia a left flank skin incision (~2.5 cm long)
was made and the spleen was exposed and pulled onto the exterior surface of the peritoneum
with blunt forceps. The splenic blood vessels were tied off with sutures and the spleen was
removed. The abdominal wall and the skin incision were closed with sutures. Sham
operations were also performed where the spleen was exteriorized and then reinserted into
the abdominal cavity.
Splenocytes were prepared from spleens of male wild-type mice by forcing the tissues
through a fine wire mesh. Cells were washed and resuspended in 2 ml Dulbeccos Phosphate
Buffered Saline (PBS) containing 10% fetal calf serum, 2 mM glutamine and 100 g/ml of
streptomycin and penicillin, and contaminating erythrocytes were lysed using 1 red cell
lysis buffer (BD Biosciences). One hour after MCAO, isolated splenocytes (2 107 in 250
ul PBS) or equal volume of PBS were infused (3 minutes) into splenectomized or shamsplenectomized mice via the femoral vein.
Drug Administration
Simvastatin (Sigma Chemicals) was prepared as a 5 mg/ml stock as previously described
[25]. Briefly, 5 mg of simvastatin was dissolved in 100 l of ethanol and 150 l of 0.1 N
NaOH, incubated at 50C for 2 h, then added water to 800 ul volume and the pH adjusted to
7 with 1N HCl, and final volume corrected to 1ml. This simvastatin stock solution was
stored at -80C and diluted with sterile saline immediately before use. Animals were
randomized to receive subcutaneous (SC) injection of simvastatin 20 mg/kg/day or saline
treatment for 5 days, with the first dose given immediately after MCAO. The doses of statin
chosen are consistent with the primary bioactive dose used in vivo in mouse/rat studies
(typically 10100 mg/kg/day) [25-27].
Brain Infarct Measurement

On day 5 after MCAO, the mice were euthanized by deep anesthesia and then perfused
transcardially with 30 ml phosphate buffered saline (PBS) followed by cold 4%
paraformaldehyde (PFA) in PBS. The brains were collected, and incubated overnight in the
4% PFA solution and then transferred into 10%, 20% and 30% sucrose solution until they
sank. Then, coronal sections (40-m thick) were serially cut using a cryostat, starting from
the frontal pole through the entire brain, and 5 coronal sections at 1 mm intervals (relative to
bregam: +1, 0, -1, -2, -3 mm) were selected and stained with 0.1% crystal violet solution
(pH 3.8) for 5 min. The areas of the uncorrected infarcted area and the total areas of both
hemispheres were measured for each slice by a computerized NIH image analysis system.
Corrected infarct area in a slice was calculated by subtracting the area of normal tissue in the
ipsilateral hemisphere from the total area of the contralateral hemisphere. Total corrected
infarct volume was then calculated by multiplying the area by the slice thickness and
summing the volumes from all slices [28]. All measurements were performed by 2 blinded
researchers and the mean of their results was calculated.
Jin et al.
Page 4
Neurological Scoring
The neurological scores were assessed by a 28-point neurological score test by an

experimenter blinded to the experimental groups at 1d, 3d and 5d. Scoring was based on
physical appearance and behavior using a scale previously developed to provide a precise
assessment of focal neurologic damage [29]. The mice were scored in each of the following
seven categories: body symmetry, gait, climbing, circling behavior, forelimb symmetry,
compulsory circling, and sensory response. Each mouse was scored from 0 (no deficit) to 4
(severely affected) in each category, as previously described [29].
Detection of Stroke-induced Splenocyte Apoptosis
Fresh spleens from sham-operated and MCAO mice were collected and whole splenocytes
were obtained by gentle grinding of spleens between frosted glass slides. The erythrocytes
were lysed with BD Pharm Lyse Lysing Buffer (Cat. No. 555899), and single cell
suspensions were collected by passing the tissue through a 100-m nylon mesh. Collected
splenocytes were washed twice in PBS, counted, and reconstituted in Ca2+-rich annexin V
binding buffer (BD Pharmingen) at a concentration of 107 cells/mL. Cell suspension (100
l) was stained with 2.5 l annexin V-FITC and/or 1 l propidium iodine (PI) for 15 min
and adjusted to a total volume of 500 l with binding buffer. Cells were then analyzed by
flow cytometry with FACSCalibur (BD Biosciences), and annexin V+/PI- cells were
considered as apoptotic cells. The percentage of apoptotic cells was calculated according to
total annexin V+/PI- divided by total cells.
Analysis of THC-induced Splenocyte Apoptosis In Vitro
Fresh spleen cells were isolated from C57BL/6 mice as described above. THC-induced
apoptosis in splenocytes was analyzed as described previously [30]. A single cell suspension
in complete medium [RPMI 1640 medium (Sigma) supplemented with 5% FBS, 10 mM
HEPES, 1 mM glutamine, 40 ug/ml gentamicin sulfate, and 50 uM 2-mercaptoethanol] was
added to 24-well flat bottom plates (1106 cells/well) and treated without or with
simvastatin (25 M) for 1h, followed by 10 M delta-9-tetrahydrocannabinol (THC, Sigma)
for additional 16h. After that, the cells were harvested and washed twice with PBS/
0.2%BSA and stained with annexin V-FITC and PI for flow cytometry analysis.
Western blot analysis
Mice were transcardially perfused with ice-cold PBS and spleens were removed and
splenocytes isolated as described above. The cytosolic and mitochondria fractions were
prepared from the isolated splenocytes using a commercially available cytosol/mitochondria
fractionation kit according to the manufacturers protocol (Millipore). Samples were stored
at -80C for further analysis. Antibodies were used as follows: monoclonal rabbit antibody
against Bcl-2 (1:1000), Bax (1:1000), COX-V (1:1000) (All purchased from Cell Signaling)
and mouse antibody against -actin (Sigma). Immunopositive bands of horseradish
peroxidase-conjugated secondary antibodies were detected with an ECL system (GE
Healthcare) and exposure to ECL Hyperfilm.
Enzyme-linked immunosorbent assay (ELISA)
Mice were transcardially perfused with ice-cold PBS and the ipsilateral cerebral cortex was
collected and immediately homogenized in five volumes of PBS with complete protease
cocktail (Roche, Indianapolis, IN, USA) using a dounce homogenizer. After incubation on
ice for 5 min, the extracts were cleared by centrifugation at 14000 r.p.m. for 10 min. The
protein content of each extract was determined by the bradford protein assay (Bio-Rad).
ELISA assays were performed using Mouse IFN- ELISA kit (Thermo scientific, Rockford,
IL, USA) following the manufacturers instructions.
Jin et al.
Page 5
Histopathological Analysis of Lung Tissue
Mice were anesthetized and intracardially perfused with 30 ml PBS followed by 20 ml of

4% PFA. Lungs were removed, postfixed in 4% PFA for overnight, and then embedded in
paraffin. 6-m thick sections were obtained by microtome dissection and stained with
hematoxylin/eosin (HE). 20 representative sections of lungs were chosen per animal and
evaluated by two investigators, who were blinded to the treatment groups. The sections were
graded according to the following criteria: no definite damage represented no histological
changes or minor changes, including unequal distension of alveolar units, mild thickening of
the alveolar septa, and perivascular and peribronchiolar edema. Definite damage was
observed as lung consolidation, thickened alveolar septae, and the presence of intra-alveolar
inflammatory infiltrates [31].
Microbiological Analysis of Blood and Lung Tissue
Mice were euthanized at indicated time points after MCAO and washed with 70% ethanol
under sterile conditions. Blood was taken by cardiac puncture after thoracotomy, and lobes
of lungs were collected, minced and homogenized under sterile conditions. Bacterial strains
from lung tissue homogenates grown on blood agar plates were examined by our Clinical
Pathology Laboratory at LSU Medical Center-Shreveport. For determination of colonyforming units (CFU), 100 L of lung tissue homogenate or blood was serially diluted in
PBS, plated onto blood agar plates (Merck), and incubated at 37 C for 18 h [31], and then
bacterial colonies were counted by two researchers blinded to treatment groups.
Statistical Analysis
Variables were analyzed by Students t-test (two-tailed) and a P<0.05 was considered
statistically significant. Values are expressed as meanSD (standard deviation) of at least
three independent experiments. Animal survival rates between the groups were analyzed
using the log-rank test.
Results
Simvastatin attenuates stroke-induced splenic atrophy and body weight loss
Splenic atrophy is an important feature of stroke-induced peripheral immunodepression and

is characterized by reduced organ size and reduced number of spleen cells that occurred
within the first few days after a stroke [20, 21]. The spleen weight and the number of
splenocytes in vehicle-treated groups were significantly decreased at 3 and 5 days after
MCAO, when compared with sham controls (Fig. 1a, b). The reduction and regain of body
weight was measured as an index of general stress elicited by cerebral ischemia. In vehicletreated animals, body weight was significantly decreased at 1 day and continued to decrease
5 days after MCAO (Fig. 1c). Simvastatin treatment significantly attenuated the splenic
atrophy and body weight loss within the 5-day observation period (Fig. 1a-c).
Simvastatin inhibits splenocyte apoptosis through mitochondrial pathway
Induction of apoptosis is one of the mechanisms leading to splenic atrophy in experimental
stroke [20, 21]. Previous studies with mice have shown that splenocyte apoptosis was
significantly induced as early as 12 h after transient MCAO [31], and the apoptosis was
further increased over time (22-96 h) after stroke [20]. Thus, we investigated whether
simvastatin reduced spleen atrophy through inhibition of splenocyte apoptosis at both the
early (12h) and late (5d) time points after MCAO. Flow cytometry analysis was performed
to identify and quantify splenocyte apoptosis. Annexin V+ PI cells are considered apoptotic
cells. The number of Annexin V+ PIapoptotic cells from the spleens of the vehicle-treated
mice was significantly increased at 12 h and further increased at 5 d after MCAO, but this
Jin et al.
Page 6
increase at both time points was significantly inhibited by simvastatin treatment (Fig. 2a,
2b). Moreover, we determined whether simvastatin can directly act on spleen cells in vitro.
The apoptosis of splenocytes isolated from C57Bl5 mice was induced by delta-9Tetrahydrocannabinol (THC), a well-known immunosuppressive agent [30]. Treatment with
THC (10 uM for 16 h) significantly increased apoptosis of splenocytes (Fig. 2c, 2d),
consistent with published data [30]. Clearly, simvastatin significantly decreased THCinduced spleen cells apoptosis in vitro (Fig. 2c, 2d).
Further, we examined the molecular mechanisms underlying the simvastatsin-mediated antiapoptotic effects. The ratio of proapoptotic Bax to anti-apoptotic Bcl-2 proteins is
considered as a major checkpoint in apoptosis [32]. The membrane and cytosolic fractions
of the spleen cells were prepared from the sham or MCAO mice treated with vehicle or
simvastatin, and the fractions were analyzed by Western blotting to measure the expression
levels of these apoptosis regulatory proteins. The expression of Bcl-2 protein in
mitochondria was markedly reduced in the vehicle-treated group, but this reduction was
largely reversed in the simvastatin-treated group (Fig. 2e). Bax is mostly cytosolic and
translocates to mitochondria following an apoptotic stimulus. The expression of Bax was
significantly decreased in the cytosol by a concomitant increase in the mitochondria of the
spleens from the vehicle-treated stroke group, but these changes were reversed by
simvastatin treatment (Fig. 2e).
Simvastatin reduces brain IFN expression and brain injury contributed by splenocytes
We performed splenectomy or sham-operation 2 weeks prior to MCAO in C57Bl6 mice.
The mean total infarct volume (5d) was 30.6 7.9 mm3 in splenectomized animals and 54.7
7.0 mm3 in sham controls (P < 0.01) (Fig. 3a). The 28-point neurological score (3d, 5d)
was also improved in splenectomized animals (Fig. 3b), compared with sham controls.
Furthermore, we demonstrated that adoptive transfer of isolated splenocytes into
splenectomized mice abolished the stroke-protective effects of splenectomy on infarct
volumes (Fig. 3a) and neurological score (Fig.3b). These data established a role of spleen in
a mouse stroke model. Moreover, simvastatin treatment significantly decreased infarct
volume (39.5 5.9 mm3) (Fig. 3a) and improved neurological score (Fig. 3b) in
splenectomized mice adoptively transferred with splenocytes. In addition, we observed that
sivastatin treatment and splenectomy did not significantly alter regional cerebral blood flow
(Suppl. Figure S1) and blood physiological parameters (Suppl. Table S1).
Next, we examined if and how simvastatin affects the IFN protein expression in the brain
at 72 h after MCAO. This time point was chosen based on the published data showing that
IFN protein levels were increased in the brain most prominently at 72h after MCAO in rats
[17]. We demonstrated that IFN protein levels were increased in the brain of shamsplenectomied MCAO mice, but the increase was abolished in splenectomied MCAO mice
(Fig. 3c). Furthermore, the IFN protein levels in the brain of splenectomied MCAO mice
were regained by adoptive transfer of isolated splenocytes (Fig. 3c), and this was largely
abolished by simvastatin treatment (Fig. 3c). In addition, simvastatin treatment significantly
reduced the IFN protein levels in the brain of MCAO mice without a splenectomy or
sham-splenectomy (Fig. 3d). Collectively, these data suggest that simvastatin protects the
brain against ischemic injury contributed by spleen cells through modulating brain IFN
expression.
Simvastatin reduces brain damage and mortality in acute experimental stroke
Previous work showed that simvastatin pre-treatment reduced brain injury at early time
point (24 h) after transient MCAO in mice [14]. We further determined whether simvastatin
protects brain against ischemic injury at a later time point after stroke. Brain infarction at 5
Jin et al.
Page 7
days after MCAO was measured on cresyl violet-stained coronal sections. Animal survival
rate from the same experimental groups was evaluated daily in the 5-day observation period.
Infarct volumes (5d) was 40.3 10.1 mm3 in the simvastatin-treated animals and 53.2 9.3
mm3 in vehicle-treated controls (P<0.05) (Fig. 4a). The 28-point neurological score (1, 3,
5d) were also significantly reduced in the simvastatin-treated animals (Fig. 4b), compared
with vehicle-treated controls. Impressively, all experimental mice (n=9) survived in the
simvastatin-treated stroke group, while only 7/13 (53.8%) of mice survived in the vehicletreated stroke group over the 5-day observation period (P<0.05) (Fig. 4c).
Simvastatin reduces lung susceptibility to spontaneous bacterial infection after stroke
Histological examination revealed typical signs of bacterial pneumonia, showing lung
consolidation, thickened alveolar septa, and intra-alveolar inflammatory infiltrates [33], in
the vehicle-treated stroke mice at 72 h after MCAO, but the damage to lung tissue was
significantly attenuated in the simvastatin-treated stroke (n=5 per group, Fig. 5a). Moreover,
lung sections of sham controls revealed no signs of pneumonia (n=4, Fig. 5a). Therefore,
susceptibility to infection resulted from stroke but not from surgical stress. In addition,
splenectomy did not increase lung susceptibility to bacterial infection 72 after MCAO (Fig.
S2).
Microbiological analysis revealed significant bacterial loads in lung cultures of all stroke
mice at 24 and 72 h after MCAO (Fig. 5b). By examining bacteria grown on blood agar
plates, we observed that bacterial cultures from lungs exhibited >95% gram-negative E. coli,
consistent with other reports [31]. In contrast, lung cultures from sham-operated mice
remained sterile at 72 h (Fig. 5b). In addition, no bacterial growth was observed in the blood
cultures of either sham or MCAO mice, consistent with previous reports in C57Bl6 mice
[31, 34]. Simvastatin significantly reduces bacterial growth in the lung tissue cultures (Fig.
5b). These data suggest that simvastatin attenuates stroke-induced lung susceptibility to
bacterial infection.
Discussion
Stroke-induced peripheral immunodepression occurs mostly during the first few days after
acute stroke and involves profound loss of immune cells, in particularly T- and B- cells, in
secondary lymphatic organs (spleen, thymus and lymph node), leading to atrophy of these
organs [19-21, 31]. The present data demonstrated a profound atrophy of spleen at 3 and 5
days of stroke in mice with a significant loss of body weight during the 5-day observation
period. Further, our work reveals the potential mechanism by which simvastatin attenuates
stroke-induced splenic atrophy, which is mediated, at least in part, through increased
expression of mitochrondrial anti-apoptotic Bcl-2 protein and decreased translocation of
proapoptotic Bax from the cytosol to mitochondria. These results should provide a
molecular basis for the beneficial immune modulatory effects of simvastatin in the
prevention of stroke-induced peripheral immunodepression.
The spleen is an important immune organ and has been implicated in the immune response
to ischemic injury in several organs (e.g. liver, lungs, intestines) [35-37]. Previous studies
have suggested that splenic atrophy in experimental stroke may contribute to brain injury
possibly through 1) release of inflammatory mediators and 2) release of spleen-derived
inflammatory cells into the circulation and migration into the brain, which exacerbate the
brain inflammatory response and cause secondary brain damage [17-21, 38]. In accordance
with this concept, splenectomy has been shown to have beneficial effects in animal models
of ischemic stroke [17, 18, 22], hemorrhagic stroke [39], and traumatic brain injury [40].
The present data demonstrated that splenectomy two weeks prior to MCAO in mice
significantly reduces infarct volume and neurological deficits 5 days after stroke. Further,
Jin et al.
Page 8

the stroke-protective effect of splenectomy was abolished by adoptive transfer of

splenocytes. These results indicate that spleen substantially contributes to brain injury in the
mouse stroke model utilized in the present study. The inflammatory signals from the spleen
to the ischemic brain have not been completely identified. Our previous data suggest that
IFN plays an important role in acute experimental stroke, as IFN knockout mice show
reduced infarct volume following transient MCAO [41]. Several studies suggest that IFN
could play a role in the splenic response by exacerbating the inflammation associated with
ischemic brain injury. Recently, Seifert et al [17, 18] reported that IFN was elevated early
(24h) in spleens but later (72, 96h) in the brains of rats following MCAO, and the
neuroprotection of splenectomy is most likely caused by the loss of IFN, because
splenectomy reduced IFN expression in the brain after MCAO and that systemic
administration of IFN reversed the protective effects of splenectomy. Moreover,
intraventricular administration of IFN neutralizing antibodies three days post-MCAO
protects the brain from stroke induced injury [42]. Collectively, these findings indicate that
IFN may be one of the inflammatory signals originating from the spleen causing a delayed
inflammatory response in the ischemic brain, supporting a role for spleen-derived IFN in
stroke pathology. In this regard, the present study focused on investigating stroke-induced
IFN expression in the brain and its modulation by adoptive transfer of splenocytes and by
simvastatin treatment. The present data suggest that stroke-induced IFN expression in the
brain is associated with the splenic response to cerebral ischemia. Simvastatin treatment
reduced brain IFN (3d) and infarct volume and neurological deficits (5d) in acute
experimental stroke, with a significant increase in animal survival over a 5-day period of
observation. These protective effects by simvastatin were observed not only in nave MCAO
stroke mice but also in splenectomied MCAO mice adoptively transferred with splenocytes.
These findings support the hypothesis that the splenic response to cerebral ischemia
contributes to secondary brain injury and simvastatin attenuates ischemic brain injury
contributed by spleen cells via modulating IFN expression in the brain in acute
experimental stroke.
Immunodepression after stroke increases the susceptibility to infections, in particular

pneumonia, the most relevant complication in stroke patients [1, 2]. In addition to
cholesterol lowering effects, statins have anti-inflammatory and immunomodulatory
properties, so called pleiotropic effects [13]. Despite some controversies exist, the treatment
of patients with statins has been shown to improve both incidence and survival in acute
ischemic stroke [13]. A recent clinical study shows that young patients with a first ischemic
stroke who used statin poststroke had lower rates of new vascular events in a long-term
follow-up [43]. However, whether statins can help to prevent stroke-induced infections is
still debated in clinical practice [13, 44, 45]. Bacterial pneumonia is the most common cause
of death in patients suffering from acute stroke and occur mostly within the first few days
after stroke [4, 5]. In the present study, we analyzed the infection status of mice in the first 3
days after acute experimental stroke. In agreement with previous reports [19, 31, 34], the
present data show that 60 min-MCAO resulted in no bacterial growth in blood samples and
bacterial growth in 100% of lung tissue cultures obtained from C57Bl6 mice. Notably,
simvastatin treatment significantly inhibited the bacterial growth in the lung tissue cultures.
It is worthy of note that the incidence of poststroke infections may vary largely depending
on not only stroke models but also mouse strains [19, 34]. Liesz et al [19] reported that mice
with pure cortical infarcts in the coagulation model had no bacterial growth in blood and
low-level bacterial growth in 30% of the lung tissue cultures; 30 min-MCAO resulted in no
bacterial growth in blood samples and bacterial growth in 50% of lung tissue cultures. In
contrast, 60% of the 90 min-MCAO mice had bacterial growth in blood cultures and 100%
had growth in lung homogenates. Moreover, Schulte-Herbrggen et al [34] reported that the
susceptibility to poststroke infections in mice is strain dependent. They compared poststroke
infections in mice from 129SV, C57/B6, and Balb/C strains subjected to a 60 min-MCAO.
Jin et al.
Page 9
Three days after stroke, 129SV mice did not only develop bacterial chest infection, but also
had a strongly increased susceptibility to bacteremia. In contrast, C57BL/6 and Balb/C mice
acquired bacterial lung infections only, and these differences in susceptibility to infection
did not correlate with infarct volumes. Therefore, careful interpretation of the results should
be used when translating these experimental findings into clinical practice.
In summary, the results of the present study provide the first direct experimental evidence
that simvastatin ameliorates stroke-induced peripheral immunodepression by attenuating
spleen atrophy and lung bacterial infection. These findings contribute to a better
understanding of beneficial effects of statins in the treatment of stroke, and also may provide
a rationale for future investigation of translational applicability for administration of
simvastatin prophylactically and/or therapeutically using clinically relevant time windows of
acute ischemic stroke. This study has some limitations, including no studies of comparisons
of simvastatins effects with other statins as well as the detailed mechanisms how statins
modulate immune response after ischemic stroke; Thus, further studies are needed.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Sources of Funding
This work was supported by National Institutes of Health grant HL087990 (GL) and HL026441 (DNG) and by a
scientist development grant (0530166N, GL) from the American Heart Association.
References
1. Dirnagl U, Klehmet J, Braun JS, Harms H, Meisel C, Ziemssen T, et al. Stroke-induced

immunodepression: experimental evidence and clinical relevance. Stroke. 2007; 38(2 Suppl):770
773. [PubMed: 17261736]
2. Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med. 2011;
17:796808. [PubMed: 21738161]
3. Brait VH, Arumugam TV, Drummond GR, Sobey CG. Importance of T lymphocytes in brain injury,
immunodeficiency, and recovery after cerebral ischemia. J Cereb Blood Flow Metab. 2012; 32:598
611. [PubMed: 22293986]
4. Westendorp WF, Nederkoorn PJ, Vermeij JD, Dijkgraaf MG, van de Beek D. Post-stroke infection:
a systematic review and meta-analysis. BMC Neurol. 2011; 11:110. [PubMed: 21933425]
5. Katzan IL, Cebul RD, Husak SH, Dawson NV, Baker DW. The effect of pneumonia on mortality
among patients hospitalized for acute stroke. Neurology. 2003; 60:620625. [PubMed: 12601102]
6. Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments.
Neuron. 2010; 67:181198. [PubMed: 20670828]
7. Bu DX, Griffin G, Lichtman AH. Mechanisms for the anti-inflammatory effects of statins. Curr
Opin Lipidol. 2011; 22:165170. [PubMed: 21412153]
8. Chen J, Zhang ZG, Li Y, Wang Y, Wang L, Jiang H, et al. Statins induce angiogenesis,
neurogenesis, and synaptogenesis after stroke. Ann Neurol. 2003; 53:743751. [PubMed:
12783420]
9. Zhang L, Zhang ZG, Ding GL, Jiang Q, Liu X, Meng H, et al. Multitargeted effects of statinenhanced thrombolytic therapy for stroke with recombinant human tissue-type plasminogen
activator in the rat. Circulation. 2005; 12:34863494. [PubMed: 16316970]
10. Lynch JR, Wang H, McGirt MJ, Floyd J, Friedman AH, Coon AL, et al. Simvastatin reduces
vasospasm after aneurysmal subarachnoid hemorrhage: Results of a pilot randomized clinical trial.
Stroke. 2005; 36:20242026. [PubMed: 16051891]
Jin et al.
Page 10

11. Jung KH, Chu K, Jeong SW, Han SY, Lee ST, Kim JY, et al. HMG-CoA reductase inhibitor,
atorvastatin, promotes sensorimotor recovery, suppressing acute inflammatory reaction after
experimental intracerebral hemorrhage. Stroke. 2004; 35:17441749. [PubMed: 15166393]
12. Seyfried D, Han Y, Lu D, Chen J, Bydon A, Chopp M. Improvement in neurological outcome after
administration of atorvastatin following experimental intracerebral hemorrhage in rats. J
Neurosurg. 2004; 101:104107. [PubMed: 15255259]
13. Montecucco F, Quercioli A, Mirabelli-Badenier M, Viviani GL, Mach F. Statins in the treatment of
acute ischemic stroke. Curr Pharm Biotechnol. 2012; 13:6876. [PubMed: 21470160]
14. Cakmak A, Yemii M, Kksoy C, Yazgan U, Diner D, Dalkara T. Statin pre-treatment protects
brain against focal cerebral ischemia in diabetic mice. J Surg Res. 2007; 138:254258. [PubMed:
17275846]
15. Zacharek A, Chen J, Cui X, Yang Y, Chopp M. Simvastatin Increases Notch Signaling Activity
and Promotes Arteriogenesis After Stroke. Stroke. 2009; 40:254260. [PubMed: 18927449]
16. Chen J, Zhang C, Jiang H, Li Y, Zhang L, Robin A, et al. Atorvastatin induction of VEGF and
bDNF promotes brain plasticity after stroke in mice. J Cereb Blood Flow Metab. 2005; 25:281
290. [PubMed: 15678129]
17. Seifert HA, Leonardo CC, Hall AA, Rowe DD, Collier LA, Benkovic SA, et al. The spleen
contributes to stroke induced neurodegeneration through interferon gamma signaling. Metab Brain
Dis. 2012; 27:131141. [PubMed: 22354752]
18. Seifert HA, Hall AA, Chapman CB, Collier LA, Willing AE, Pennypacker KR. A transient
decrease in spleen size following stroke corresponds to splenocyte release into systemic
circulation. J Neuroimmune Pharmacol. 2012; 7:10171024. [PubMed: 23054371]
19. Liesz A, Hagmann S, Zschoche C, Adamek J, Zhou W, Sun L, et al. The spectrum of systemic
immune alterations after murine focal ischemia: immunodepression versus immunomodulation.
Stroke. 2009; 40:28492858. [PubMed: 19443795]
20. Offner H, Subramanian S, Parker SM, Wang C, Afentoulis ME, Lewis A, et al. Splenic atrophy in
experimental stroke is accompanied by increased regulatory T cells and circulating macrophages. J
Immunol. 2006; 176:65236531. [PubMed: 16709809]
21. Offner H, Subramanian S, Parker SM, Afentoulis ME, Vandenbark AA, Hurn PD. Experimental
stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow
Metab. 2006; 26:654665. [PubMed: 16121126]
22. Ajmo CT Jr, Vernon DO, Collier L, Hall AA, Garbuzova-Davis S, Willing A, et al. The spleen
contributes to stroke-induced neurodegeneration. Neurosci Res. 2008; 86:22272234.
23. Jin R, Song Z, Yu S, Piazza A, Nanda A, Penninger JM, et al. Phosphatidylinositol-3-kinase
gamma plays a central role in blood-brain barrier dysfunction in acute experimental stroke. Stroke.
2011; 42:20332044. [PubMed: 21546487]
24. Coligan JE. Current Protocols in Immunology. 1991:1.10.11.10.11.
25. Leung BP, Sattar N, Crilly A, Prach M, McCarey DW, Payne H, et al. A novel anti-inflammatory
role for simvastatin in inflammatory arthritis. J Immunol. 2003; 170:15241530. [PubMed:
12538717]
26. Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, et al. Stimulation of bone formation in
vitro and in rodents by statins. Science. 1999; 286:19461949. [PubMed: 10583956]
27. Dimmeler SA, Aicher M, Vasa C, Mildner-Rihm K, Adler M, Tiemann H, et al. HMG-CoA
reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway.
J Clin Invest. 2001; 08:391397. [PubMed: 11489932]
28. Takano K, Tatlisumak T, Formato JE, Carano RA, Bergmann AG, Pullan LM. Glycine site
antagonist attenuates infarct size in experimental focal ischemia. Postmortem and diffusion
mapping studies. Stroke. 1997; 28:12551262. [PubMed: 9183359]
29. Doyle KP, Yang T, Lessov NS, Ciesielski TM, Stevens SL, Simon RP, et al. Nasal administration
of osteopontin peptide mimetics confers neuroprotection in stroke. J Cereb Blood Flow Metab.
2008; 28:12351248. [PubMed: 18364727]
30. McKallip RJ, Lombard C, Martin BR, Nagarkatti M, Nagarkatti PS. Delta(9)tetrahydrocannabinol-induced apoptosis in the thymus and spleen as a mechanism of
Jin et al.
Page 11

immunosuppression in vitro and in vivo. J Pharmacol Exp Ther. 2002; 302:451465. [PubMed:
12130702]
31. Prass K, Meisel C, Hoflich C, Braun J, Halle E, Wolf T, et al. Stroke-induced immunodeficiency
promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by
poststroke t helper cell type 1-like immunostimulation. J Exp Med. 2003; 198:725736. [PubMed:
12939340]
32. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in
apoptosis. Genes Dev. 1999; 13:18991911. [PubMed: 10444588]
33. Wang Q, Teder P, Judd NP, Noble PW, Doerschuk CM. CD44 deficiency leads to enhanced
neutrophil migration and lung injury in Escherichia coli pneumonia in mice. Am J Pathol. 2002;
61:22192228. [PubMed: 12466136]
34. Schulte-Herbrggen O, Klehmet J, Quarcoo D, Meisel C, Meisel A. Mouse strains differ in their
susceptibility to poststroke infections. Neuroimmunomodulation. 2006; 13:1318. [PubMed:
16612133]
35. Okuaki Y, Miyazaki H, Zeniya M, Ishikawa T, Ohkawa Y, Tsuno S, et al. Splenectomy reduced
hepatic injury induced by ischemia/reperfusion in the rat. Liver. 1996; 16:188194. [PubMed:
8873006]
36. Savas MC, Ozguner M, Ozguner IF, Delibas N. Splenectomy attenuates intestinal ischemiareperfusion-induced acute lung injury. J Pediatr Surg. 2003; 38:14651470. [PubMed: 14577069]
37. Jiang H, Meng F, Li W, Tong L, Qiao H, Sun X. Splenectomy ameliorates acute multiple organ
damage induced by liver warm ischemia reperfusion in rats. Surgery. 2007; 141:3240. [PubMed:
17188165]
38. Bao Y, Kim E, Bhosle S, Mehta H, Cho S. A role for spleen monocytes in post-ischemic brain
inflammation and injury. J Neuroinflammation. 2010; 7:92. [PubMed: 21159187]
39. Lee ST, Chu K, Jung KH, Kim SJ, Kim DH, Kang KM, et al. Anti-inflammatory mechanism of
intravascular neural stem cell transplantation in haemorrhagic stroke. Brain. 2008; 131(Pt 3):616
629. [PubMed: 18156155]
40. Li M, Li F, Luo C, Shan Y, Zhang L, Qian Z, et al. Immediate Splenectomy Decreases Mortality
and Improves Cognitive Function of Rats After Severe Traumatic Brain Injury. J Trauma. 2011;
71:141147. [PubMed: 21248654]
41. Yilmaz G, Arumugam TV, Stokes KY, Granger DN. Role of T lymphocytes and interferon-gamma
in ischemic stroke. Circulation. 2006; 113:21052112. [PubMed: 16636173]
42. Liesz A, Suri-Payer E, Veltkamp C, Doerr H, Sommer C, Rivest S, et al. Regulatory T cells are
key cerebroprotective immunomodulators in acute experimental stroke. Nat Med. 2009; 15:192
199. [PubMed: 19169263]
43. Putaala J, Haapaniemi E, Kaste M, Tatlisumak T. Statins after ischemic stroke of undetermined
etiology in young adults. Neurology. 2011; 77:426430. [PubMed: 21810695]
44. van den Hoek HL, Bos WJ, de Boer A, van de Garde EM. Statins and prevention of infections:
systematic review and meta-analysis of data from large randomised placebo controlled trials. BMJ.
2011; 343:d7281. [PubMed: 22127443]
45. Yeh PS, Lin HJ, Chen PS, Lin SH, Wang WM, Yang CM, et al. Effect of statin treatment on threemonth outcomes in patients with stroke-associated infection: a prospective cohort study. Eur J
Neurol. 2012; 19:689695. [PubMed: 22176026]
Jin et al.
Page 12

Jin et al.
Page 13

Jin et al.
Page 14

Figure 1. Simvastatin attenuates stroke-induced splenic atrophy and body weight loss
A, Quantification of spleen weights and cell numbers in sham control mice and MCAO mice
treated with vehicle or simvastatin. B, Representative pictures of spleens in sham and
MCAO mice at the indicated time points of MCAO. Scale bar = 2 mm. C, Quantification of
body weight loss in the indicated groups. A & C: n=7-9 mice per group per time point; ##
P<0.01 vs. respective sham control; * P<0.05, **P<0.01 vs. respective vehicle control at the
same time point.

Jin et al.
Page 15

Jin et al.
Page 16

Jin et al.
Page 17

Jin et al.
Page 18

Figure. 2. Simvastatin attenuates splenocyte apoptosis through mitochondrial pathway
A, B Mice were euthanized 12 hours or 5 days after MCAO for splenic analysis. A,
Representative flow cytometric dot plots for FITC-Annexin V and propidium iodine (PI)
labeling in freshly isolated splenocytes from sham control mice and MCAO mice treated
with vehicle or simvastatin. Annexin V+ and PI- cells were considered early apoptotic cells
(lower right quadrant). B, Flow cytometric analysis of stroke-induced splenocyte apoptosis
in the simvastatin- and vehicle- treated mice at the indicated time points after MCAO. The
percentage of apoptotic cells was calculated from the ratio of apoptotic cells to total cells
counted. Data represent the mean SD of four independent experiments. # P<0.05 vs. sham
control; * P<0.05 vs. vehicle control. C, Representative flow cytometric dot plots showing
THC-induced splenocyte apoptosis in vitro. D, Flow cytometric analysis of THC-induced
splenocyte apoptosis in vitro. Data represent the mean SD of five independent
experiments. #P<0.05 vs. Vehicle; *P<0.05 vs. the THC only group. E, Western blot
analysis of the protein levels of mitochondrial Bcl-2, mitochondrial and cytosolic Bax in the
splenic cells from sham control mice and MCAO mice treated with vehicle or simvastatin.
-actin and COX IV were used as loading controls for cytosolic and mitochondrial fractions,
respectively. Data are presented as mean SD of four independent experiments (n=4 mice/
group for each experiment). * P<0.05, ** P<0.01 vs. sham control; # P<0.05 vs. vehicle
control.
Jin et al.
Page 19

Jin et al.
Page 20

Jin et al.
Page 21

Jin et al.
Page 22

Figure 3. Simvastatin reduces brain IFN protein expression and brain damage contributed by
splenic cells
Splenectomy and adoptive transfer of splenocytes were performed as described in the

Method. Stroke analysis was performed 5 days after MCAO. A, Representative images of
cresyl violet-stained coronal brain sections (upper) and Quantitative analysis of infarct
volumes (lower). Scale bar = 2 mm. B, The 28-point neurological scoring at indicated time
points (n= 6-8 survived mice/group). *P<0.05 vs. sham-splenectomized MCAO
mice; #P<0.05 vs. splenectomized MCAO mice, and &P<0.05 vs. splenectomized MCAO
mice with adoptive transfer of splenic cells (SC) at the same time point. C and D, ELISA
measurement of IFN- protein concentrations in the ipsilateral hemispheres from the
indicated groups at 72h after MCAO. n=5 mice/group.
Jin et al.
Page 23

Jin et al.
Page 24

Figure 4. Simvastatin reduces ischemic brain injury and animal mortality 5 days after MCAO
Jin et al.
Page 25
A, Representative images of cresyl violet-stained coronal brain sections (left panels) and
Quantitative analysis of infarct volumes (right panels). Scale bar = 2 mm. B, The 28-point
neurological scoring at indicated time points in the vehicle-treated and simvastatin-treated
mice. n = 7-9 per group. *P<0.05 vs. vehicle-treated group at the same time point. C,
Animal survival rate from the same experiments was recorded daily for 5 days after MCAO.
n=9 for simvastatin- and n=13 for vehicle-treated mice. *p<0.05, log-rank test compared
with vehicle-treated mice.

Jin et al.
Page 26

Figure 5. Simvastatin attenuates stroke-induced lung susceptibility to spontaneous bacterial

infection
A, Representative images of HE-stained lung sections (6-m thick) showing typical signs
(thickening of alveolar walls and neutrophilic infiltrates) of pneumonia from MCAO (72h)
but not from sham animals. Scale bar = 50 m. B, Lungs and blood samples from sham and
MCAO mice treated with vehicle or simvastatin were prepared for bacteriological analysis
at the indicated time points after surgery. Data are expressed as CFU/ml (log 10) in blood or
lung tissue homogenate. N=3-6 per group. # P<0.05 vs. vehicle control at 4h after MCAO; *
P<0.05 vs. vehicle control at same time point.

Simvastatin Spleen Atropi

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Simvastatin Spleen Atropi

Uploaded by

Copyright:

Available Formats

NIH Public Access

NIH-PA Author Manuscript

Published in final edited form as:

Simvastatin attenuates stroke-induced splenic atrophy and lung

NIH-PA Author Manuscript

NIH-PA Author Manuscript

ConclusionsResults provide the first direct experimental evidence that simvastatin

NIH-PA Author Manuscript

Statins are inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)

NIH-PA Author Manuscript

Post-stroke peripheral immunodepression is characterized by profound atrophy of secondary

NIH-PA Author Manuscript

procedures. In ischemic groups, animals were subjected to 60 minutes MCAO followed by

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Brain Infarct Measurement

NIH-PA Author Manuscript

The neurological scores were assessed by a 28-point neurological score test by an

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Histopathological Analysis of Lung Tissue

NIH-PA Author Manuscript

Mice were anesthetized and intracardially perfused with 30 ml PBS followed by 20 ml of

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Splenic atrophy is an important feature of stroke-induced peripheral immunodepression and

Stroke. Author manuscript; available in PMC 2014 April 01.

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Stroke. Author manuscript; available in PMC 2014 April 01.

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

the stroke-protective effect of splenectomy was abolished by adoptive transfer of

NIH-PA Author Manuscript

Immunodepression after stroke increases the susceptibility to infections, in particular

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

1. Dirnagl U, Klehmet J, Braun JS, Harms H, Meisel C, Ziemssen T, et al. Stroke-induced

Stroke. Author manuscript; available in PMC 2014 April 01.

NIH-PA Author Manuscript

Stroke. Author manuscript; available in PMC 2014 April 01.

NIH-PA Author Manuscript

Stroke. Author manuscript; available in PMC 2014 April 01.

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Splenectomy and adoptive transfer of splenocytes were performed as described in the

Stroke. Author manuscript; available in PMC 2014 April 01.

NIH-PA Author Manuscript